Inhibition of Rhabdomyosarcoma’s Bone Marrow Metastasis through Blocking of MET Receptor Signaling.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma belonging to family of small blue cell tumors and it represents the largest subset of soft tissue sarcomas in this age group. RMS can be divided into two major subtypes based on chromosomal analysis - embryonal (ERMS) without translocations and alveolar (ARMS) with translocations. They differ in clinical outcome with ERMS having more favorable prognosis. However, nevertheless of subtype, if RMS is diagnosed at late stage (IV) or with metastatic disease the outcome is usually poor with only 21% of 5 years overall survival. RMS gives distant metastasis to various organs but predominantly to lung and bone marrow. Recently, MET receptor, which is highly expressed on RMS cells, has been shown to mediate RMS cells motility. Since HGF, a ligand for MET receptor, is abundantly expressed by bone marrow cells, it was also postulated that MET could be involved in bone marrow metastasis of RMS (Jankowski et al. Cancer Research 2003). In this study we have shown that blocking of MET receptor expression by RNA interference (RNAi) has strong influence on metastatic behavior of RMS cells both in vitro and in vivo. Using RNAi we created both ERMS and ARMS cell lines that lack MET receptor expression as was assessed by qRT-PCR and Western blotting. We showed in vitro that these cells possessed a reduced potential to migrate toward HGF (as study by chemotaxis, invasion and “would healing’ assays) and to secrete extracellular matrix degrading enzymes after stimulation with HGF. However, we did not see any influence of MET downregulation on mitogenic potential of RMS cells. We also found that inhibition of HGF-MET signaling blocks adhesion of RMS to stromal and endothelial cells. Most importantly, inactivation of HGF-MET axis strongly suppressed the ability of RMS cells to engraft and populate bone marrow cavities both in short and long term in vivo assays. In 24 hour seeding assay all mice injected i.v. with wild type RMS had detectable tumor cells in their bone marrow as measured by qRT-PCR and flow cytometry. At the same time mice injected with MET negative cells had bone marrow cavities free of tumor. In long term assay mice were injected s.c. with 5×10⁶ RMS and after 30 days presence of tumor cells in bone marrow was investigated. qRT-PCR analysis revealed that 100% of mice injected with wild type cells and only 50% of mice injected with RMS negative cells had metastatic tumor in their bone marrow. In summary, we conclude that MET receptor plays a very important role in facilitating metastasis of RMS into bone marrow and that blocking of HGF-MET axis could be used in the future as a therapeutic option to inhibit spreading of RMS into this organ.